Identification of quantitative trait loci governing arthritis severity and humoral responses in the murine model of Lyme disease.

A spectrum of disease severity has been observed in patients with Lyme disease, with approximately 60% of untreated individuals developing arthritis. The murine model of Lyme disease has provided strong evidence that the genetic composition of the host influences the severity of arthritis following infection with Borrelia burgdorferi: infected C3H mice develop severe arthritis while infected C57BL/6N mice develop mild arthritis. Regions of the mouse genome controlling arthritis severity and humoral responses during B. burgdorferi infection were identified in the F2 intercross generation of C3H/HeNCr and C57BL/6NCr mice. Rear ankle swelling measurements identified quantitative trait loci (QTL) on chromosomes 4 and 5, while histopathological scoring identified QTL on a unique region of chromosome 5 and on chromosome 11. The identification of QTL unique for ankle swelling or histopathological severity suggests that processes under distinct genetic control are responsible for these two manifestations of Lyme arthritis. Additional QTL that control the levels of circulating Igs induced by B. burgdorferi infection were identified on chromosomes 6, 9, 11, 12, and 17. Interestingly, the magnitude of the humoral response was not correlated with the severity of arthritis in infected F2 mice. This work defines several genetic loci that regulate either the severity of arthritis or the magnitude of humoral responses to B. burgdorferi infection in mice, with implications toward understanding the host-pathogen interactions involved in disease development.

Analysis of antibody response to the outer surface protein family in lyme borreliosis patients.

Studies on frequencies of serum antibodies to outer surface proteins (Osps) in Lyme disease have produced conflicting results. Osp antigens (A, B, and C) enriched by butanol extraction, which aids band identification in immunoblotting, were used to test sera for IgG antibody to Osp antigens from Borrelia burgdorferi isolates from each subspecies (sensu stricto, afzelii, and garinii). Individual isolates were selected to include all five known European OspA genotypes. Of arthritis sera, 83% (n=29), and of acrodermatitis chronica atrophicans sera, 81% (n=26), recognized OspA, B, and/or C. Of erythema migrans sera, 23% recognized OspA and/or B and a further 15% OspC alone. Only 5 (6%) of 86 sera (4 arthritis, 1 acrodermatitis chronica atrophicans, 0 erythema migrans) recognized all five OspA phenotypes tested. Marked differences in the reactions of individual sera to the various Osp antigens were seen, which helps reveal the causes of discrepancies between previous reports.

Specific Th1 cell lines that confer protective immunity against experimental Borrelia burgdorferi infection in mice.

Although humoral responses to Borrelia burgdorferi (Bb) have been shown to be protective in some animal models of Lyme disease, the role of T cells in this disease is less well understood. This work describes three Bb-specific T cell lines that prevent disease progression in syngeneic mice. The T cell lines were generated in C3H mice immunized with Bb in complete Freund's adjuvant. All lines were Bb-specific, CD4+, TCRalphabeta+, and they proliferated and produced interferon-gamma and interleukin-2 on stimulation with Bb. Injection of the cell lines into naive C3H recipients significantly reduced the number of organisms recoverable from the blood and tissues of infected mice and protected them from developing Bb-induced periarthritis. These studies demonstrated that Th1 cells can confer resistance to Bb infection in susceptible mice and suggested that the timing of this T cell response may be critical for determining disease outcome.

Infectivity and arthritis induction of Borrelia japonica on SCID mice and immune competent mice: possible role of galactosylceramide binding activity on initiation of infection.

We investigated the relationship between the binding activity to galactosylceramide (GalCer) and the arthritis induction activity of Borrelia japonica. The B. japonica strains maintained the ability to induce arthritis in inbred C3H/HeN and immunodeficient SCID mice, but the ability was lower than that of Borrelia burgdorferi sensu stricto virulent strain 297. Histopathological changes were restricted to the joints, and a marked effusion of polymorphonuclear neutrophils into the joint space was found. The binding activity of B. japonica strains to GalCer was lower than that of the virulent strain 297 but higher than that of the high-passage strain 297. The lower infectivity and virulence of B. japonica may explain its lower binding ability to GalCer.

Characterization of the physiological requirements for the bactericidal effects of a monoclonal antibody to OspB of Borrelia burgdorferi by confocal microscopy.

A confocal microscopy study was undertaken to characterize the bactericidal effects of the Fab fragments of CB2, an immunoglobulin G1kappa murine monoclonal antibody, to an epitope in the carboxy region of the outer surface protein B (OspB) of Borrelia burgdorferi. Simultaneous direct labeling of both fixed and live spirochetes with fluorochrome-labeled Fab-CB2 and 11G1, and an immunoglobulin Mkappa monoclonal antibody to OspA, showed that OspA and OspB seem to colocalize in dead spirochetes but do not appear to be physically associated when the organisms are alive. A polar bleb composed of a Fab-CB2-OspB complex, followed by incorporation of 11G1-OspA, precedes the formation of a spheroplast. The spheroplasts contain both OspA and OspB and are a terminal stage in the bactericidal process induced by Fab-CB2. Outer membrane destabilization by Fab-CB2, but not cell wall or cytoplasmic membrane alterations, was demonstrated experimentally by the sequential treatment of spirochetes with Fab-CB2 and monoclonal antibodies to flagellin and DnaK. The action of Fab-CB2 is epitope specific, as another monoclonal antibody to an epitope in the amino terminus of OspB was not bactericidal. The bactericidal effect of Fab-CB2 is not dependent on the induction of spirochetal proteases but is dependent on the presence of Ca2+ and Mg2+. Supplementation of Ca2(+)- and Mg2(+)-free medium with these cations restored the bactericidal effects of Fab-CB2. The mechanism by which a Fab fragment of an antibody destroys a bacterium directly may represent a novel form of antibody-organism interaction.

Analysis of the human antibody response to outer surface protein C (OspC) of Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii.

The aim of this study was to determine by Western blotting (WB) the prevalence of anti-outer surface protein C (OspC) IgM and IgG antibodies in patients with Lyme borreliosis according to each of the three genospecies of Borrelia burgdorferi sensu lato. Strains of B. burgdorferi sensu stricto (MUL), B. garinii (DK 6), and B. afzelii (DK 26) served as antigen, all of which expressed abundant OspC. We examined sera from 117 patients with untreated early and late Lyme borreliosis, as well as from 100 blood donors and 29 patients with syphilis. WB results were compared with the B. burgdorferi flagellum enzyme-linked immunosorbent assay (ELISA) data. OspC from B. burgdorferi sensu stricto showed the lowest diagnostic sensitivity. OspC from B. garinii and B. afzelii performed almost identically in erythema migrans, with an IgM positive rate of 36% versus 34%, whereas OspC from B. garinii performed best in neuroborreliosis (60% versus 44%). The anti-OspC IgG response was less prominent than the IgM response and was infrequent in the late stages of the disease (0-20%). The benefit of combining the evaluation of anti-OspC responses with all three species was limited. The overall diagnostic sensitivity of WB anti-B. garinii OspC evaluation was, in the early stages of the disease, comparable to the results obtained using the flagellum ELISA. In erythema migrans and neuroborreliosis, the addition of anti-OspC IgM to the flagellum ELISA increased the sensitivity by 15% and 10%, respectively. It can, therefore, be concluded that OspC from B. garinii is a suitable OspC test antigen, and that supplementary use of OspC from other species adds little to the overall diagnostic sensitivity. An ELISA based on B. garinii OspC and native flagella seems currently the most promising concept for a future antibody test in early Lyme borreliosis.

Variant responses of mice to Borrelia burgdorferi depending on the site of intradermal inoculation.

C3H/He mice inoculated intradermally at one of two sites with Borrelia burgdorferi responded differently to infection. Shoulder-inoculated mice developed spirochetemia, B. burgdorferi-specific antibody, and arthritis earlier than foot-inoculated mice. Lymphocyte populations derived from spleen tissue were elevated in the shoulder- but not the foot-inoculated mice, and those from lymph nodes were increased in both groups. Lymphocytes derived from blood and spleen tissue showed impaired proliferative responses to all mitogens for shoulder-inoculated mice only, whereas proliferation of lymph node cells was not affected, regardless of route. These results demonstrate that the site of initial B. burgdorferi inoculation is an important determinant in the pathogenesis of B. burgdorferi infection.

Efficacy of clarithromycin for treatment of experimental Lyme disease in vivo.

Clarithromycin provided effective therapy against arthritis induced by Borrelia burgdorferi infection in the hamster. In vitro, clarithromycin was at least 1 log more potent than tetracycline against two isolates of B. burgdorferi from human sources, as measured by MICs and 50% inhibitory concentrations. Clarithromycin was effective in preventing the onset of B. burgdorferi-induced arthritis, as determined by several parameters of paw swelling. When administered after the onset of arthritis, clarithromycin therapy reduced the degree of swelling and decreased recovery time. These results suggest that clarithromycin has potential as an effective therapy for Lyme disease.

Pathogenetic role of Chlamydia, Yersinia and Borrelia in undifferentiated oligoarthritis.

We studied the cellular and humoral immune response to Chlamydia trachomatis, Yersinia enterocolitica and Borrelia burgdorferi in paired samples of peripheral blood and synovial fluid (SF) in undifferentiated oligoarthritis, reactive arthritis (ReA) and rheumatoid arthritis. Antigen specific lymphocyte proliferation was found in SF of 43% of patients with ReA and 34% of patients with undifferentiated oligoarthritis. C. trachomatis was the most frequent single agent. HLA-B27 was positive in 83% of patients with ReA and in 62% of patients with undifferentiated oligoarthritis with antigen specific lymphocyte proliferation. Antigen specific lymphocyte proliferation correlated poorly with the specific antibody response. Only chlamydial antigen was detected in SF cells using monoclonal antibodies. We conclude that some patients with undifferentiated oligoarthritis may have a forme fruste of ReA. This finding is important in view of recent evidence supporting the efficacy of antibiotic therapy in ReA.

[Suspected Lyme borreliosis: how to diagnose, whom to treat?]. / Verdacht auf Lyme-Borreliose: Wie abklären, wen behandeln?

Lyme Borreliosis is diagnosed or presumed primarily on a clinical basis. Serological tests may help to increase the diagnostic certainty. Type and duration of antibiotic therapy depend on the stage of the disease.

Seroepidemiological survey for antibody to Borrelia burgdorferi in cows.

Antibody to Borrelia burgdorferi was examined in 380 healthy and 38 clinical cases of cows from Hokkaido and Shizuoka in Japan. In healthy animals, IgG and IgM antibody to B. burgdorferi HO14 strain were found in 44 cows (14.6%) and 24 cows (8.0%) from Hokkaido. In contrast, antibody-positive case was not observed except for only 1 case which was IgM positive (1/79: 1.3%) in cows from Shizuoka. Mean antibody levels of healthy animals in Hokkaido and Shizuoka were 0.651 and 0.263 (IgG antibody to HO14 strain), 0.642 and 0.169 (IgG to HP3 strain), 0.613 and 0.367 (IgM to HO14 strain) and 0.582 and 0.286 (IgM to HP3 strain). The differences of the antibody levels between cows from Hokkaido and Shizuoka were significant. Seasonal difference was found in seropositive cows from Hokkaido. The rate of seropositive cows was high in summer (23.4% in June and 11.8% in July) but low in winter (0% in January and February). The pattern was discussed to be associated with activation of ticks. One of 4 cows with arthritis showed significantly higher IgG antibody level than that of healthy cows and cows with some disease, although the serum was collected from Shizuoka where antibody-positive animals for B. burgdorferi were rare among healthy cows. This high IgG antibody may suggest that the arthritis of such cows was caused by infection with B. burgdorferi. Two of 7 cows with unclassified abortion showed positive antibody reaction in Hokkaido. These cases, however, may not be related to the B. burgdorferi infection because the positive rate was similar to those of healthy cows in the same season.

Evaluation of granulocytic ehrlichiosis in dogs of Missouri, including serologic status to Ehrlichia canis, Ehrlichia equi and Borrelia burgdorferi.

Canine granulocytic ehrlichiosis was diagnosed in 37 dogs by finding ehrlichial morulae in 0.1 to 26.2% of their blood neutrophils and eosinophils. All 37 dogs had clinical signs of arthritis or muscular stiffness. Titer to Ehrlichia canis was determined in sera from 31 of the 37 dogs; 25 dogs had titer ranging from 1:20 to 1:5,120. In the other 6 dogs, titer to E canis was less than 1:10. The most common hematologic abnormality in these dogs, other than rickettsiemia, was thrombocytopenia. Granulocytes infected with ehrlichial organisms were not found in another 10 dogs that had clinical signs of arthritis or muscular stiffness. Of these 10 dogs, 3 had titer to E canis ranging from 1:40 to 1:320. Titer in the other 7 dogs was less than 1:10. Ehrlichial morulae were not found in the granulocytes of 18 healthy dogs. Of these 18 dogs, 9 had titer to E canis ranging from 1:20 to 1:5,120. Titer in the other 9 dogs was less than 1:10 Titer to Borrelia burgdorferi was determined in dogs with granulocytic ehrlichiosis, arthritic dogs without detected rickettsiemia, and in healthy dogs. Low titer determined by 2 laboratories was considered to be nonspecific reaction in all 3 groups of dogs and, thus, did not indicate that the arthritic disorders were attributable to canine borreliosis.

Experimental Borrelia burgdorferi infection in inbred mouse strains: antibody response and association of H-2 genes with resistance and susceptibility to development of arthritis.

We have investigated the specific humoral immune response and its correlation to the development of disease after experimental inoculation of B. burgdorferi in different inbred strains of mice. All mouse strains tested showed high levels of specific IgM antibodies during the initial 10 days of infection. Specific IgG antibodies predominantly of the IgG2a, IgG2b and IgG3 isotypes were found in increasing amounts by 14 days post infection. Antibody titers peaked at days 65 and 110. Particularly low titers of specific IgM and/or IgG antibodies were detected in sera of AKR/N and B10.BR mice. Antibodies specific for numerous B. burgdorferi antigens including the outer surface proteins A (31 kDa) and B (34 kDa) and a protein(s) of molecular mass of approximately 40 kDa, most probably 41 kDa (flagellin) and/or 39 kDa (p39), were induced in all inbred mouse strains within 2 weeks inoculation albeit in varying concentrations. Later during infection, the patterns of antibody specificities were much more complex. With regard to development of disease all strains of mice tested fall into three groups: (a) mice of H-2k haplotype (AKR/N, C3H/HeJ, C3H/HeN, B10.BR) developed a chronic progressive arthritis in the tibiotarsal joints, (b) mice of H-2 haplotypes, H-2b (C57BL/6), H-2j (B10.WB), H-2r (B10.R111) and H-2s (B10.S) developed arthritis of variable duration and intensity which was not progressive and (c) mice of H-2d haplotype (BALB/c, DBA/2, C.B-17, B10.D2, Cal.20), irrespective of their background genes or Igh allotype, showed no clinical signs of arthritis at any time point following inoculation of B. burgdorferi organisms. The finding of similar patterns of apparently protective antibodies in all mouse strains tested together with the striking association between the H-2d haplotype and resistance, and between the H-2k haplotype and the occurrence of B. burgdorferi-induced arthritis suggest a critical role of T cells in the development of the disease in mice.

[Lyme disease with the triad of neurologic manifestations].

A 40-year-old man developed progressive neurologic manifestations following a tick bite with subsequent localized erythema migrans. These manifestations included bilateral sensory radiculoneuritis (T7-12), rectovesical dysfunction, paraparesis, right facial palsy and nuchal rigidity. Both serum and cerebrospinal fluid titers of IgG antibody against Borrelia burgdorferi were 1:8,192 using indirect immunofluorescence assay. No IgM antibody was detected. With high-dose intravenous penicillin and corticosteroid treatment the neurologic symptoms and signs gradually subsided, with a corresponding decrease in the IgG antibody titers. Among the cases of Lyme disease reported so far in Japan, the present case seems to be typical and serious in terms of the severity of nervous system involvement and the intensity of the antibody response.

Ceftriaxone therapy of chronic inflammatory arthritis. A double-blind placebo controlled trial.

To determine whether chronic inflammatory arthritis may respond to antibiotic therapy (implying a bacterial origin), we conducted a placebo-controlled, double-blind study. Sixty patients with inflammatory arthritis and antibody titers to Borrelia burgdorferi 1:64 or more were randomized to receive placebo (n = 20) or 2 g/d of ceftriaxone intravenously (n = 40) for 2 weeks. Two of 20 placebo- and 19 of 40 antibiotic-treated patients improved. At 1 month, the placebo-treated patients could elect to receive ceftriaxone. Altogether, 58 patients were treated with ceftriaxone and followed up for 13 to 24 months. Improvement was noted in 27 of the 58 antibiotic-treated patients. Patients with a wide diversity of inflammatory arthritis were studied. Response to ceftriaxone was seen in all groups, including 5 of 12 with rheumatoid arthritis, 5 of 8 with psoriatic arthritis, 3 of 5 with vasculitis, and 14 of 33 with less well-differentiated chronic inflammatory arthritis. In 16 of the 27 who responded to the antibiotic, the arthritis worsened 6 to 18 months after the initial response to ceftriaxone. Previous improvement of arthritis after oral antibiotic was a better predictor of response to ceftriaxone than either duration of disease or Lyme antibody titer. Side effects to ceftriaxone were frequent and included diarrhea (29/60) and acute allergic reactions (9/58). We conclude that some patients may have an occult bacterial infection underlying their chronic inflammatory arthritis, and may respond to antibiotic therapy. The response to ceftriaxone in patients with even weakly reactive Lyme titers encourages further prospective placebo-controlled studies of antibiotics in various subsets of chronic arthritis.

Cefotaxime versus penicillin in the late stage of Lyme disease--prospective, randomized therapeutic study.

The low responsiveness of Lyme arthritis to high dose intravenous penicillin G therapy has evoked the demand for new drugs for the treatment of late stage borreliosis. As can be deduced from in vitro susceptibility data, third generation cephalosporins are far more effective on Borrelia burgdorferi spirochetes than penicillin G. The study presented here was designed to compare cefotaxime at a dosage of 2 x 3 g/day with penicillin G at a dosage of 2 x 10 megaunits/day, for ten days in a prospective randomized trial. A total of 135 patients were included in the study. They were diagnosed to suffer from late stage Lyme borreliosis on the basis of defined clinical symptoms compatible with stage three borreliosis manifestations of at least six months' duration and positive antibody titers against B. burgdorferi. Final outcomes were recorded after a 24 month post-treatment observation period with re-examination at three-month-intervals. Cefotaxime proved to be significantly superior to penicillin G with 87.9% versus 61.3% of treatments resulting in full or incomplete remission of symptoms (p = 0.002). Clinical remission was accompanied by declining antibody titers. Herxheimer-like reactions were observed in 20% of the patients of the penicillin group and in 40.5% of the patients of the cefotaxime group and may be interpreted as an indication of a response to therapy.